Peroxidase
Peroxidases are an ubiquitous family of enzymes found in many living organisms, including plants. Many peroxidases are glycoproteins with a heme group located at the active site that oxidize molecules at the expense of hydrogen peroxide. The median molecular weight of these proteins is in the range of 40 to 50 kDa and numerous isoenzymes are known to exist in a given organism.
Peroxidases can be generally divided into anionic, cationic and neutral enzymes. The basis of this distinction is their isoelectric points and probable functions. Cationic isoenzymes (pI 8-11) in the central vacuole catalyze the synthesis of H.sub.2 O.sub.2 from NADH and H.sub.2 O and act as an indole acetic acid oxidase. Neutral isoenzymes (pI 4.6-6.5) on cell walls have activity with lignin precursors and possibly function in wound healing. Strongly anionic (pI 3.5-4) peroxidases function in lignificiation.
The plant peroxidase isoenzymes may have roles in a wide variety of functions in physiology and development. These roles include auxin catabolism, cross-linking of phenolic components of the cell wall, and suberin and lignin biosynthesis (Gaspar, et al., Peroxidases 1970-1980: A Survey of their Biochemical and Physiological Roles in Higher Plants. University of Geneva, Switzerland, pp. 1-324, 1982; Fry, Ann. Rev. Plant. Physiol., 37: 165-186, 1986; Mohan and Kolattukudy, Plant Physiol., 92: 276-280, 1990; Gross, Adv. Bot. Res. 8, 26-65, 1980). Peroxidases are induced by wounding and are presumably involved in the repair and formation of cell walls. Peroxidase has been shown to catalyze the polymerization of phenolic compounds into lignin and form cross-links between extensin, a hydroxyproline rich cell wall protein and lignin, and feruloylated polysacchrides (Reviewed by Grisebach, The Biochemistry of Plants, 7: 457-478, 1981). They have been also implicated in the regulation of cell wall elongation (Goldberg, et al., in Molecular and Physiological Aspects of Plant Peroxidases, Geneva, Switzerland: University of Geneva, pp. 209-220, 1986).
In cotton, peroxidase is located in ovary walls that becomes the carpel tissue of the boll. Peroxidase is also associated with developing ovules and fibers (Mellon, Plant Cell Rep., 5: 338-341, 1986; Wise and Morrison, Phytochemistry, 10: 2355-2359, 1971).
Genetic Engineering of Cotton
Although successful transformation and regeneration techniques have been demonstrated in model plants species such as tobacco (Barton, et al., Cell, 32: 1033-1043, 1983), similar results with cotton have only been achieved relatively recently. See, e.g. Umbeck, et al. Bio/Technology, 53! 263-266 (1987); Firoozabady, et al., Plant Mol. Bio., 10: 105-116 (1987); Finer and McMullen., Plant Cell Rep., 8: 586-589, 1990. McCabe and Martinell Bio/Technology, 11: 596-598, 1993.
Cotton is one of the most important cash crops. Successful transformation and regeneration of genetically engineered cotton plants has the potential to be of significant value to this agriculturally important crop. One of the most important benefits potentially achievable from genetically engineering cotton plants is the alteration and modification of cotton fiber quantity and quality.
Cotton fiber (seed hair) is a differentiated single epidermal cell of the ovule. At maturity the fiber cell consists of a cell lumen, primary cell-wall and secondary cell-wall. The primary cell-wall is made up of pectic compounds, cellulose, and small amounts of protein. The secondary cell-wall consists of cellulose. At maturity, the cotton fiber contains 87% cellulose.
Cotton fiber development can be divided into initiation, primary cell-wall synthesis stage, secondary cell-wall deposition stage, and maturation phases. Many hundreds of genes are required for the differentiation and development of cotton fiber. Work on in vitro translated fiber proteins (Delmer, et al., J. Cell Sci., 2: 33-50, 1985) and protein isolated from fiber (Graves and Stewart, J. Exp. Bot., 39: 59-69, 1988) clearly suggests differential gene expression during various developmental stages of the cell. Only a few of the genes involved in the biosynthesis of the large numbers of fiber-specific structural proteins, enzymes, polysaccharides, waxes or lignins have been identified (John and Crow, Proc. Natl. Acad. Sci. USA, 89: 5769-5773, 1992). Since these genes and their interactions with environment determine the quality of fiber, their identification and characterization is considered to be an important aspect of cotton crop improvement.
The quality of the cotton fiber is dependent on such factors as the extent of elongation and degree of secondary wall deposition. It is assumed that both a number of genes and environmental factors regulate the physical characteristics of the fiber such as length, strength and micronaire value. However, the genes responsible for cellulose synthesis and fiber development in cotton plants are heretofore entirely uncharacterized at a molecular level.
The most commercially useful plant fiber is derived from cotton (Gossypium arboreum, Gossypium herbaceum, Gossypium barbadense and Gossypium hirsutum). However, there are other fiber-producing plants with potential commercial uses. These plants include the silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca and flax.
Promoters
Promoters are DNA elements that direct the transcription of RNA from DNA sequences. Together with other regulatory elements that specify tissue and temporal specificity of gene expression, promoters control the development of organisms. Thus, there has been a concerted effort in identifying and isolating promoters from a wide variety of plants and animals.
Many promoters function properly in heterologous systems. For example, promoters taken from plant genes such as rbcS, Cab, chalcone synthase and protease inhibitor from tobacco and Arabidopsis are functional in heterologous transgenic plants. (Reviewed by Benfey and Chua, Science, 244: 174-181, 1989). Specific examples of transgenic plants include tissue-specific and developmentally regulated expression of soybean 7s seed storage protein gene in transgenic tobacco plants (Chen, et al. EMBO J., 7: 297-302, 1988.) and light-dependent organ-specific expression of Arabidopsis thaliana chlorophyll a/b binding protein gene promoter in transgenic tobacco (Ha and An, Proc. Natl. Acad. Sci. USA, 85: 8017-8021, 1988). Similarly, anaerobically inducible maize sucrose synthase-1 promoter activity was demonstrated in transgenic tobacco (Yang and Russell, Proc. Natl. Acad. Sci. USA, 87: 4144-4148, 1990). Tomato pollen promoters were found to direct tissue-specific and developmentally regulated gene expression in transgenic Arabidopsis and tobacco (Twell, et al., Development, 109: 705-714, 1990). Similarly, one cotton promoter has been shown to express a transgene in a fiber-specific manner (John and Crow, Proc. Natl. Acad. Sci. USA, 89: 5769-5773, 1992). Thus, some plant promoters can be utilized to express foreign proteins in specific tissues in a developmentally regulated fashion.
Many of the features of over-expression of peroxidases could be advantageously combined with plant fiber. Because of the potential for debilitating effects on normal plant physiology by over-production of highly catalytic enzymes such as peroxidase, we believe that it is preferable to confine expression of these enzymes to limited tissues. This strategy allows for normal plant growth and the specific effect of over-expression of the gene in specific tissue, such as fiber. However, a fiber-producing plant containing a tissue-specific peroxidase genetic construct has neither been proposed nor created. What is needed in the art of molecular biology is a plant fiber containing over-expression of peroxidase.